Phytochemical Analysis and Anti-microbial Activity of Some Important Medicinal Plants from North-west of Iran

Due to the increase of microbial resistance to antibiotics and the occurrence of side effects, use of medicinal plants with anti-microbial properties seems to be rational. Hence, in this study, some plants of the Apiaceae, Asteraceae, Brassicaceae, and Cucurbitaceae families were evaluated for antimicrobial effects. The aerial parts of the plants were extracted by different solvents using a Soxhlet apparatus. Subsequently, the inhibitory effect of the extracts on different microbial species was assessed. Extracts with high growth inhibitory effect were fractionated and their MIC was determined. Furthermore, primary phytochemical and GC-MS analysis were used to identify the chemical compounds of potent samples of n-hexane extracts of Eryngium caerulum (E. caeruleum) and Eryngium thyrsoideum (E. thyrsoideum.) Both plants showed considerable antimicrobial activities against Staphylococcus epidermidis among the fractions, 40% and 60% VLC fractions of n-hex extract of E. caeruleum and 40% VLC fraction of n-hexane extract of E. thyrsoideum illustrated the most growth inhibitory effect. Moreover, the results of preliminary phytochemical and GC-MS analysis confirmed that steroids, fatty acids and terpenoids play an important role to show anti-microbial activity, respectively. Among all samples, the 40% VLC fraction of n-hexane extract of E. thyrsoideum for possessing high amounts of fatty acids and terpenoids indicated the most anti-microbial potency.


Introduction
Infectious illnesses are recognized as one of the significant leading sources of morbidity and mortality in the world (1). The anti-microbial compounds which can retard microbial growth or cause microbial mortality with minimum toxicity to host cells are deliberated campaign for promoting anti-microbial substances.
Furthermore, due to the growing increase of multiple antibiotic resistance in pathogenic microorganisms in the last two decades from all over the world and also to the development of adverse effects of main antibiotics and the occurrence of previously unusual infectious diseases, a search for new plants with antimicrobial properties to determine new natural antibiotics which might decrease mentioned complications (2-6). Herbal medicines are the main sources of natural drugs which can be a substitute to the common medicines (7,8). Recently, anti-microbial properties of herbal drugs are being progressively identified in the most parts of the world (9)(10)(11)(12)(13)(14)(15)(16). In this research project, the anti-microbial activities of some plants traditionally used by people of Iran, were screened against different microbial species including gram positives, gram negatives, and a fungi. Moreover, preliminary qualitative phytochemical as well as GC-MS analysis of potent extracts and fractions have been illustrated which phytochemicals are responsible for showing antimicrobial effect. The plants, examined in this study, were generally used to cure the infections and related symptoms (Table  1). In this regard, we report the conclusion of this study for future research planning in order to find effective and non-toxic anti-microbial medicines.

Plant material
Seven species of 4 authenticated plant families used in the traditional medicine by Iranian people were collected from different regions of Iran. All of the plants, after identification by a senior herbal taxonomist, Dr. Atefeh Ebrahimi, have been deposited at the herbarium of the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.

Preparation of plant extracts and fractions
Dried powdered aerial parts of Antriscus nemorosa, Artemisia marschalliana, Eryngium caeruleum, Eryngium thyrsoideum, Lepidium vesicarium and also the seeds of Eryngium elaterium were extracted by soxhlet apparatus using n-hexane (n-hex), dichloromethane (DCM), and methanol (MeOH) (1.1 L each, Caledon company, Canada). The filtered extracts were separately dried and concentrated by using a rotary evaporator vacuum at 45 °C. Dried extracts and fractions were diluted in DMSO to prepare the stock concentration 100 mg mL -1 for anti-microbial tests.

Antimicrobial bioassay Test microorganisms
Anti-microbial activity of plant extracts

Disk diffusion method
In this method, Muller-Hinton-agar plates were cultured with a standard 0.5 McFarland (10 8 CFU/mL) inoculum of the test microorganisms. Then 4 filter paper discs (about 6 mm in diameter) and one standard antibiotic disk (positive control) were placed on the surface of each inoculated plate. Forty microliter of each extract solution was transferred over 3 blank disks and one disk was impregnated with DMSO as a negative control. In the next step, they were incubated in 37 °C for 24 h. Finally, for evaluating the anti-microbial activity, the diameter of inhibition zone against the tested organisms was measured by a caliper (16).

Minimal Inhibitory Concentration (MIC) determination
For determining the MIC of potent extracts and their fractions, modified broth microdilution method was used. In this test, 100 μL distilled water was added to each well of a sterile 96-well microtitre plate and then 100 μL extract solution (100 mg mL -1 ) was added to first well and was diluted two-fold serially until eighth well. Afterwards, 100 μL of each bacterial suspension (5 × 10 5 CFU/mL) in Muller Hinton Broth (MHB) medium was added until eight well. Three control wells were filled with 1) DMSO and culture medium containing bacterial suspension, 2) culture medium containing bacterial suspension only and 3) culture medium with plant extract, respectively. The final volume in each well was 200 μL. After well-mixing, the microtitre plate was incubated for 24 h. Finally, the lowest concentration that totally inhibited the growth of microorganism was determined as the MIC and the test was repeated three times (17).

Fractionation of non-polar extracts
For further investigations, the potent extracts (inhibited strongly growth of the bacteria) were subjected to Vacuum Liquid Chromatography (VLC) method. VLC is a method to obtain different fractions of non-polar extracts (18,19). In this method, first VLC hopper was connected to Buchner filter, and then a filter paper was put on the filter. In the next step, the silica gel was loaded into the 2/3 of the tightened hopper. Subsequently, the vacuum pomp was used to compact the silica gel. Another filter paper was placed on the silica gel column. After these preparations, 150 mL methanol, 150 mL ethyl acetate and 150 mL ethyl acetate 10% (10% Et OAc and 90% n-hex) were passed over the silica gel, respectively. Then, the filter paper over the column was removed. Afterwards, n-hex extract was solved in adequate EtOAc 10% and then the solution was loaded over the column and then step by step several concentrations of Et OAc in n-hex (10%, 20%, 40%, 60%, 80% and 100%) were passed through the column and were collected in separate containers. In the end, the pure methanol passed over the silica gel to purify the column.

Preliminary phytochemical analysis Phytochemical analysis A: Steroids and Triterpenoids tests
Libermann-Buchard test: little amount of acetic anhydride were added to the different samples and mixed together. Subsequently, by adding the concentrated H 2 SO 4 slowly, brown ring at the gap of two layers was observed through one hour. The color changes in up and down layer were used to distinguish the steroids from triterpenoids. Formation of bluish green and red color in upper and down layer was considered for the presence of steroids and triterpenoids, respectively (16).

B: Tests for Glycosides Libermann-Buchard test
The Libermann-Buchard test as the primary test proved the presence of Steroids and Triterpenoids. Correspondingly, Kedd and keller killiani tests as main characteristic of the cardiac glycoside Components, were applied for determining the existence of unsaturated lactones and deoxy sugars, respectively.
Kedd΄s test 2-3 drops of (3, 5 dinitro benzoic acid) 2%, in 90% alcohol were added to dry samples, then the PH of Kedde΄s reagent with 20% KOH was fixed in alkali range. Formation of purple color indicated the presence of β-unsaturated -O-lactones.

Keller-killiani test
The mixture of CH 3 COOH and FeCl 3 was added to the dried samples. Following, by adding the concentrated H 2 SO 4 , the color changes were observed to bluish green and reddish in upper and down layers, respectively. It is better to add the sulfuric acid slowly by the flank of the sample tube.

C: Tests for Alkaloids
Dragendorff's test Brownish precipitate was observed by adding the potassium bismuth iodide solution to the samples (Dragendorff reagent).

Hagerʹs test
The test specimens which contain alkaloids show yellow precipitate in presence of Hager reagent (Saturated solution of picric acid).

D: Test for Tannins
Dark green and blue color appeared in the samples which have tannins, by adding the 5% Ferric chloride.

E: Test for flavonoids Shinoda test
Amount of HCL 37% with all test samples was mixed, Magnesium ribbon was added for accelarating the speed of color changes to red.

G: Test for Carbohydrate Benedict's test
The blending of the test samples and Benedict's reagent was boiled in water bath. Subsequently, brownish precipitation in presence carbohydrates was observed (16).

GC-MS analysis
GC/MS analysis of the potent extracts and fractions of potent samples was carried out on a Shimadzu GCMS-QP5050A gas chromatograph-mass spectrometer (GC-MS) fitted with a fused methyl silicon DB-1 column (60 m × 0.25 mm i.d., 0.25 μm film thickness). Carrier gas was helium with a flow rate of 1.3. The column temperature was programmed at 100 °C for 2 min and then increased to 300 °C at a rate of 4 °C/min increase, and finally kept constant at 300 °C for 15 min. The injector temperature was 270 °C and split ratio was set up at1:19 . The injection volume was 1 mL. The mass operating parameters were obtained at the following conditions: ionization potential 70 eV; ion source temperature 260 °C; quadrupole temperature 100 °C; solvent delay 2.0 min; resolution 2000 amu/sec, and scan range 30-600 amu; EM voltage 3000 volts.
Components of the potent samples (extracts and fractions) were identified by standard compounds, and computer matching with the NIST 107, NIST 21, NIST 69, and Wiley 229 mass spectral database, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (16). For quantitation (area%), the GC analysis was also performed using an Agilent 6890 gas chromatograph equipped with a FID detector. The FID detector temperature was 300 ºC.
To obtain the same elution with GC/MS, duplicatation of the same column and same operational conditions was applied. Relative percentage amounts of peak areas were calculated from FID chromatograms.

Results
The results of disk diffusion method and MIC determination were illustrated in Tables 2 and 3. According to Table 2, the results of screening are promising, among the extracts, 12 extracts illustrated anti-microbial activity against one or more bacteria while 2 extracts (methanolic and n-hexane extracts of A. nemorosa and L. vesicarium respectively) indicated anti-candida potency. Among the all extracts (Table 2), only the n-hex extracts of E. caeruleum and E. thyrsoideum caused significant anti-microbial effects on S. aureus and S. epidermidis. In the cases of VLC fractions of n-hex extracts ( Table  Table 2. Antimicrobial activity of plant extracts using agar disc diffusion method.

Plant
Extracts Diameter of Inhibition zone (DIZ, mm)

S.a S.e B.s L.m E.c S.t C.a
A. nemorosa      3), 60% and 40% VLC fractions of E. caerulum and also 40% VLC fraction in E. thyrsoideum against S. epidermidis (MIC = 1.562, 1.562 and 0.39 mg mL -1 , respectively) exhibited stronger biological activity, respectively. Furthermore, preliminary phytochemical tests were analyzed on total n-hex extracts in both species, which showed that the existence of common phytocomponents such as steroids play the major role to indicate the growth inhibition effect. GC-MS analysis of potent extract and its fractions in both potent species (Tables 4 and 5) showed various compounds.

Discussion
Microorganisms are considered as the mainstay and origin of multiple diseases. Germs have been identified as infectious agents in many diseases. Most commonly agents used as antimicrobial drugs have lost their effectiveness in therapeutic doses against microorganisms, and with increasing doses, the side effects of drugs increase and endanger human life, so this is a major concern of these days in the world (20). According to recent studies, naturally occurring compounds such as plants can be a good solution to this problem. In various scenarios, enormous interests in searching for new anti-microbial compounds from plants which can overcome to drug resistance have been actuated (21,22). The present study was leaded to examine the in-vitro antimicrobial activity of some plants (twenty-four extracts of 8 species of plants from 4 families) used in traditional medicine of Iran against four gram positives, two gram negatives and a fungi to investigate their advantages compared with common antibiotics. The disk diffusion and modified broth micro-dilution methods were applied for determining the anti-microbial activity of different concentrations of extracts and fractions. Presence or absence of inhibition zone indicates the potency of various kinds of extracts. Five plants including A. nemorosa, A. marschalliana, E. caeruleum, E. thyrsoideum and L. vesicarium illustrated broad scale of anti-microbial activity. The same reports on anti-microbial effects of Iranian medicinal plants such as essential oil of the root of Anthriscus nemorosa, silver nanoparticles of Artemisia marschalliana, fruits of Ecballium elaterium, essential oil of aerial parts of Eryngium caeruleum, and ethanolic extracts of Lepidium vesicarium, were also evaluated, and demonstrated different results in comparison to our work with varying degrees of potency (23-31). In our study, the sensitivity tested microorganisms was, in decreasing order: S. epidermidis, S. aureus, B. subtilis, L. monocytogenes, C. albicans, E. coli, and S. typhi. As a whole, different extracts of the plants were effective on gram-positive bacteria and among these strains, S. epidermidis and S. aureus were more affected by several extracts. Furthermore, MeOH extract of A. nemorosa and n-hex extract of L. vesicarium were effective on C. albicans, as opposed to the rest of the extracts. None of the extracts had any effect on gram-negative bacteria. In the case of the tested bacteria, it has been mentioned that the possible cause of the difference in bacterial susceptibility is the presence of an outer membrane around the cell wall of the gram-negative bacteria that prevents the release of the extract through the lipopolysaccharide coating (32). In other words, differences between the ingredients of gram positive and gram negative cell walls were as a main cause for showing different susceptibility. Moreover, in the periplasmic space of gram negative species, there are enzymes degrading external molecules (33). Among the gram positive bacteria, B. subtilis has showed minimum sensitivity in comparison to other species, which may be due to its capability to form endospores as an enormous resistant material. All of the gram negative species were more resistant upon the samples. The n-hex extracts of E. caeruleum and E. thyrsoideum caused significant anti-microbial effects on S. aureus and S. epidermidis. In the case of E. caeruleum the inhibition activity against S. aureus and S. epidermidis was approximately the same. However, S. epidermidis in comparison to S. aureus was more affected by E. thyrsoideum. S. aureus for having broad range of distribution on normal body flora, showed resistance against many drugs in comparison to S. epidermidis (34). In order to further investigation and identify anti-microbial compounds, different fractions of potent extracts (n-hex) of two plants (E. thyrsoideum and E. caeruleum) were obtained by VLC method using different percentages of EtOAc and n-hex, and also their anti-microbial activity was measured against more sensitive strains. 60% and 40% VLC fractions in E. caeruleum and 40% fraction in E. thyrsoideum both against S. epidermidis exhibited significant effect, respectively. However, MIC amount of n-hex extract of E. caeruleum in comparison to VLC fractions is low; in the case of E. thyrosoideum this quantity is vice versa. Hence, it is concluded that VLC fractions of E. caeruleum showed their inhibitory effect synergistically, but in the E. thyrosoideum main antimicrobial compounds have been accumulated in 40% VLC fractions. In many other species of Eryngium, anti-microbial activities have been investigated, but their findings have been completely different with ours. Although, in different studies, various species of Eryngium inhibited gram positive strains (35, 36), in Bazzaz and et al., research E. billardieri indicated a considerable activity against gram negative strain (E. coli). In our current assay, it did not show considerable growth inhibition effect on studied microbial strains (37). In spite of our finding (ineffectiveness of MeOH extract), in Marčetić et al.'s essay, MeOH extract of E. palmatum exerted potent effect on both gram positive and negative species (38). The differences in potency may be due to the different sensitivity of the test strains along with method of extraction. Hence, in order to determine the main differences, phytochemical analysis of n-hex extract, 40% and 60% VLC fractions of E. caeruleum as well as n-hex extract and 40% VLC fraction of E. thyrsoideum as potent agents were investigated by GC-MS analysis. Furthermore, preliminary phytochemical tests in both species showed that steroids play the major antimicrobial role. Our results in some extent are in consistent with the reports of the other researchers (36, 38 and 39). On the other hand, in n-hex extracts and 40%, 60% VLC fractions in potent species (E. caeruleum and E. thyrosoideum) fatty acids and derivatives, hydrocarbons and terpenoids were observed as the most common active compounds in a large part of volatile components, correspondingly. In the case of E. thyrosoideum, the main compounds were hydrocarbons and fatty acids. Furthermore, the presence of some steroids, terpenoids and potent anti-microbial agents like stigmasterol methyl ether, campesterol, spathulenol, limonen-6-ol, has caused the 40% VLC fraction showing considerable anti -microbial activity with minimum IC 50 . The anti-microbial effect of some above mentioned secondary metabolites have been proved by many literatures (40-46). Furthermore, different previous investigations have confirmed the anti -microbial activity of steroids and fatty acids (40, 44 and 47-52). It seems that steroids and fatty acids exert their anti-microbial potency by several mechanisms on cell membrane: Fatty acids create interim or perpetual hole on cell membrane and finally damage it for having amphipathic structure and detergent characteristic. Moreover, they can inhibit the activity of enzymes, ruin the nutrient absorption and finally can eradicate the existence of microbes by producing the free radicals (43, 47, 52 and 53). Furthermore, the findings of the previous studies speculated that the probable mechanism of anti-microbial action of terpenoids might result in the change of membrane penetrating and in permeation of intracellular agents (54, 55). Our anti-microbial screening findings affirm the traditional uses of some studied plants in different ailments containing infectious diseases.

Conclusion
To sum up, among all the studied samples, n-hex extracts, 40%, 60% VLC fractions of E. caeruleum and 40% VLC fractions of E. thyrosoideum illustrated potent anti-microbial activities against S. aureus and S. epidermidis strains. Subsequently, their chemical analysis revealed potent anti-microbial agents. It is suggested to isolate the pure compounds of potent fractions and to elucidate their structure and to do anti-microbial tests on the purified compounds.